Low dead volume extraction column device

ABSTRACT

The invention provides extraction columns for the purification of an analyte (e.g., a biological macromolecule, such as a peptide, protein or nucleic acid) from a sample solution, as well as methods for making and using such columns. The invention is characterized by the use of low dead volume columns, which is achieved in part by the use of low pore volume frits (e.g., membrane screens) to contain a bed of extraction media in the column. Low dead volume facilitates the elution of the captured analyte into a very small volume of desorption solution, allowing for the preparation of low volume samples containing relatively high concentrations of analyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S.Provisional Patent Application Serial No. 60/396,595, filed Jul. 16,2002 and U.S. Provisional Patent Application Serial No. 60/465,606,filed Apr. 25, 2003, the disclosures of which are incorporated herein byreference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] This invention relates to a device and method for the capture ofanalytes by solid phase extraction with a column device and collectionof the analytes into a controlled volume of solvent. The analytes caninclude biomolecules, particularly biological macromolecules such asproteins and peptides. The device and method of this invention areparticularly useful in proteomics for sample preparation and analysiswith analytical technologies employing biochips, mass spectrometry andother instrumentation.

BACKGROUND OF THE INVENTION

[0003] Proteomics can be defined as the comprehensive study of proteinsand their functional aspects. Proteins perform the work of the cell.Single proteins can have many forms. The function of a protein dependson the form, interactions, and complexes of the protein. A deeperunderstanding of the biological functions of proteins is needed so thatdrugs can be developed.

[0004] Protein sample processing is a complex problem within proteomics.Proteins can function individually or as complexes (groups of proteinsbound as a complex). Proteins cannot be amplified, as DNA is amplifiedwith polymerase chain reaction (PCR) methods. Proteins must be enrichedand purified before they can be analyzed. Protein processing methods andsystems must be flexible; more than a million possible proteins areexpressed. For analysis it is necessary to separate and concentrate theproteins of interest from many thousands of other proteins, whileselectively removing other materials that will interfere with theprotein analytical process including cellular material such as otherproteins, sugars, carbohydrates, lipids, DNA, RNA and salts.Reproducible recovery is needed and in most cases protein function mustbe retained during processing. Structural differences between forms mustbe preserved and final processing of samples must be easily integratedinto many different detection schemes, for example mass spectrometry,protein chips, and the like.

[0005] Solid phase extraction is one of the primary tools for preparingprotein samples prior to analysis. The method purifies proteinsaccording to their identity, class type or structure, or function toprepare them for analysis by mass spectrometry or other analyticalmethods.

[0006] The process of solid phase extraction uses an extraction phase inthe form of a column or bed, and the sample may be either loaded ontothe column or added to a bulk solution to extraction beads. Theextraction phase retains the sample protein, the extraction phase iswashed to remove contaminants, and then the sample protein is removedwith the extraction or recovery solvent.

[0007] Extraction columns are used to prepare the protein samples foranalysis. Often very low amounts of proteins are expressed in a sample,and sample preparation procedures are needed to isolate and recover theprotein before analysis.

[0008] The solid phase extraction of biomolecules such as nucleic acidsand proteins is commonly performed by columns packed with a variety ofextraction phases.

[0009] The need for biomolecule extraction for proteins is increasingrapidly. Large numbers of samples need to be analyzed by a variety oftechniques to determine the function of proteins. Typical sample volumeis 0.5 to 5 mL or more on a typical column bed volume of 1 to 5 mL,requiring a typical desorption solvent volume of 2 to 10 mL.

[0010] There are a number of companies that have developed productswhose principle aim is the purification of certain proteins or proteinclasses by solid phase extraction. The intent of these products is thesimplification of proteomic analyses by providing a sample of only thoseproteins in which the investigator is interested. These products areoften packaged for a single use and disposal. Packed-bed columns operateat relatively low pressures, thus making them simple to operate in ahighly parallel and automated manner. Due to the very nature of aconventional packed-bed approach, it is limited with respect to reliablequantification and/or enrichment of sample. A packed-bed approach isextremely difficult to apply in a manner that is both cost-effective andreliable. It cannot be effectively applied to a microscale processlevel.

[0011] Moreover, packed columns have extensive carry-over from sample tosample, are expensive to manufacture, and may be difficult to multiplex(extract multiple samples simultaneously). Proteins may be irreversiblyadsorbed to the extraction phase or may be trapped by frits and other“dead zones” within the column making recovery of the proteinsincomplete.

[0012] Other drawbacks include losses of materials due to unsweptvolumes leading to low recoveries and irreproducibility of results;dilution of materials due to large elution volumes applied in an attemptto minimize these selfsame unswept volumes; depending on implementation,requirements often to adhere to a flow “directionality” introducinglimitations on full integration of sample processing; manufacturingdifficulties and costs for micro- or nanoscale volume systems; andporosity of construction materials used in commercially availablesystems that cause severe loss of biomaterials.

[0013] Spin columns and pipette tip columns are disposable columntechnologies commonly used for processing samples. At present, most ofthese columns contain filters or frits. Conventional frits, porous discsused to contain the column beds, have significant dead volume. Thisleads to significant sample loss when very small sample volumes areseparated.

[0014] One conventional method for making sample preparation devicesinvolves first inserting a precut porous plug obtained from, forexample, a fiberous glass or cellulose sheet, into the tip of a pipette.This is followed by the addition of loose particles and a second porousplug. The plugs serve to retain the particles in place in the pipettetip. However, the plugs also entrap excess liquid thereby creating deadspace or volume (i.e., space not occupied by media or polymer that canlead to poor sample recovery, contamination such as by samplecarry-over, etc.).

[0015] Current available methods are not well suited for the separationand recovery of very small volumes in the low microliter range.

[0016] Also, since the volume of the filter is often as large as thevolume of the micro volume sample itself, the extraction or separationprocess or chromatography process is adversely affected due to the largevolume of filter material through which the sample must pass.

[0017] In addition, the adsorption of biomolecules can be a problem.Since the concentration of biomolecules in micro volume samples is sosmall, the adsorption of biomolecules on the filter can result insignificant loss of the total sample mass. The filter material may alsoabsorb proteins or biomolecules from the sample, resulting in lower thandesirable sample recovery. Also, the filter material may behavedifferently in different elution media, subsequently interfering withboth the quality of the separation process and the volume of the sampleretained.

[0018] Collecting samples in the 1 to 20 μL range is a critical need. Atsuch low volumes, efficient sample handling is crucial to avoid loss.Conventional methods and devices for sample preparation are notpractical for handling the “microseparation” of such small samplevolumes.

[0019] Ultrafiltration can only effectively concentrate and desalt, andthus the application of adsorption technology at this scale could offeran entirely new approach to micro-mass sample preparation.

[0020] However, these procedures cannot be used with extremely smallliquid delivery devices such as conventional pipette tips, as there isno practical way to load either the plug or the particles to obtain amicro-adsorptive device that contains 20 milligrams or less ofadsorbent, the amount suitable for use with the aforementioned extremelysmall sample loads.

SUMMARY OF THE INVENTION

[0021] This invention provides a column device for capturing a componentpresent in a fluid comprising a column body having a column body fittedon a pipette, syringe or similar pump for drawing liquid into and out ofthe opposite column end, and a column bed of packing material with thebed contained by two low dead volume membrane screens, the top membranescreen placed directly on top of the column bed, and the bottom membranescreen extended across the tip of the lower end of the column.

[0022] In one embodiment the invention provides a low dead volumeextraction column comprising: a column body having an open upper end forattachment to a pump, an open lower end for passing fluid into and outof the column body, and an open channel between the upper and lower endof the column body; a bottom frit bonded to and extending across theopen channel, the bottom frit having a low pore volume; a top fritbonded to and extending across the open channel between the bottom fritand the open upper end of the column body, the top frit having a lowpore volume, wherein the top frit, bottom frit, and channel surfacedefine an extraction media chamber; and a bed of extraction mediapositioned inside the extraction media chamber.

[0023] In some embodiments the bottom frit is located at the open lowerend of the column body.

[0024] In some embodiments the bottom frit is less than 200 micronsthick, has a pore volume equal to 10% or less of the interstitial volumeof the bed of extraction media an/or has a pore volume of 0.5microliters or less.

[0025] In some embodiments the extraction media comprises a packed bedof gel-type packing material, e.g, a gel-type chromatography bead. Inpreferred embodiment bed is based on agarose and sepharose.

[0026] In some embodiments the bed of extraction media has a bed volumeof less than 20 microliters.

[0027] In some embodiments the bottom frit is a membrane screen and thetop frit is optionally a membrane screen. The membrane screen cancomprise a nylon or polyester woven membrane.

[0028] In some embodiments the extraction media comprises an affinitybinding group having an affinity for a biological molecule of interest,e.g., Protein A, Protein G and an immobilized metal.

[0029] In some embodiments the column body comprises a polycarbonate,polypropylene or polyethylene material. The frits can be attached to thecolumn body by any of a variety of approaches, including by means of anannular pip, friction fit, gluing or welding.

[0030] In some embodiments the volume of the extraction media chamber isless than 20 microliters.

[0031] In some embodiments the bed of extraction media has a dry weightof less than 2 mgs.

[0032] In some embodiments the extraction media comprise an extractionbead selected from the group consisting of affinity beads used forprotein purification, ion exchange beads used for protein purification,hydrophobic interaction beads used for protein purification, reversephase beads used for nucleic acid or protein purification, agaroseprotein G beads used for IgG protein purification, and Hypercell beadsused for IgG protein purification.

[0033] In some embodiments the column body comprises a luer adapter, asyringe or a pipette tip.

[0034] In some embodiments the upper end of the column body is attachedto a pump for aspirating fluid through the lower end of the column body.

[0035] In some embodiments the pump is a pipettor, a syringe, aperistaltic pump, an electrokinetic pump, or an induction based fluidicspump.

[0036] In some embodiments the low dead volume extraction columncomprises: a lower tubular member comprising the lower end of the columnbody, a first engaging end, and a lower open channel between the lowerend of the column body and the first engaging end; and an upper tubularmember comprising the upper end of the column body, a second engagingend, and an upper open channel between the upper end of the column bodyand the second engaging end, the top membrane screen of the extractioncolumn bonded to and extending across the upper open channel at thesecond engaging end; wherein the first engaging end engages the secondengaging end to form a sealing engagement. In some of these embodimentsthe first engaging end has an inner diameter that matches the externaldiameter of the second engaging end, and the first engaging end receivesthe second engaging end in a telescoping relation. Some of theseembodiments have a first engaging end has a tapered bore that matches atapered external surface of the second engaging end.

[0037] The invention also provides a method for extracting an analytefrom a sample solution using an extraction column of the inventionwherein the upper end of the column body is attached to a pump foraspirating fluid through the lower end of the column body. The methodcomprises the steps of: contacting the lower end of the column body ofthe extraction column with a sample solution containing an analyte andaspirating a quantity of the sample solution into the column, wherebythe quantity of sample solution enters the bed of extraction media andthe analyte is adsorbed by the extraction media; discharging the samplesolution out through the lower end of the extraction column body;contacting the lower end of the column body with a desorption solventand aspirating a quantity of the desorption solvent into the column,whereby the quantity of sample desorption enters the bed of extractionmedia and the analyte is desorbed from the extraction media into thedesorption solvent; and discharging the analyte-containing desorptionsolvent out through the lower end of the column.

[0038] In some embodiments of the method, the column is attached to apump for aspirating and discharging fluid through the lower end of thecolumn body and the pump is used to discharge the sample solution andanalyte-containing desorption solvent from the extraction column.

[0039] In some embodiments of the method, a quantity of wash fluid isaspirated into the column through the lower end of the column and thendischarged out through the lower end of the column between the steps ofdischarging the sample solution out through the lower end of theextraction column body and contacting the lower end of the column bodywith a desorption solvent and aspirating a quantity of the desorptionsolvent into the column, thereby washing the bed of extraction media.

[0040] In some embodiments of the method, the volume of desorptionsolvent aspirated into the column is less than 3-fold greater theinterstitial volume of the packed bed of extraction beads.

[0041] In some embodiments of the method, the quantity of desorptionsolvent is aspirated and discharged from the column more than once.

[0042] In some embodiments of the method, the analyte is a biologicalmacromolecule, e.g, a protein. The protein-containing desorption solventcan is in some cases introduced onto a protein chip or introduced into amass spectrometer.

[0043] In some embodiments of the method, the sample solution is ahybridoma cell culture supernatant.

BRIEF DESCRIPTION OF THE FIGURES

[0044]FIG. 1 depicts an embodiment of the invention where the extractioncolumn body is constructed from a tapered pipette tip.

[0045]FIG. 2 is an enlarged view of the extraction column of FIG. 1.

[0046]FIG. 3 depicts an embodiment of the invention where the extractioncolumn is constructed from two cylindrical members.

[0047]FIG. 4 depicts a syringe pump embodiment of the invention with acylindrical bed of solid phase media in the tip.

[0048]FIG. 5. is an enlarged view of the extraction column element ofthe syringe pump embodiment of FIG. 4.

[0049] FIGS. 6-10 show successive stages in the construction of theembodiment depicted in FIGS. 1 and 2.

[0050]FIG. 11 depicts an embodiment of the invention with a straightconnection configuration as described in Example 8.

[0051]FIG. 12 depicts an embodiment of the invention with an end cap andretainer ring configuration as described in Example 9.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0052] This invention is used for the capture of analytes by solid phaseextraction with a column device and collection of the analytes into acontrolled volume of solvent. This invention is useful for analytesincluding biomolecules and is compatible with requirements for samplepreparation and analysis by analytical technology—especially biochipsand mass spectrometry.

[0053] The invention is characterized by the use of extraction columnshaving low dead volumes. This is achieved in part by the use of a lowvolume frit or frits to contain a bed of extraction media in anextraction media chamber positioned in the column column. Low deadvolume facilitates the elution of the captured analyte into a very smallvolume of desorption solution, allowing for the preparation of lowvolume samples containing relatively high concentrations of analyte. Lowvolume, high concentration solutions particularly useful with regard toprotein preparations for analysis by techniques such as massspectrometery and protein chips.

[0054] I. Terminology

[0055] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific embodimentsdescribed herein. It is also to be understood that the terminology usedherein for the purpose of describing particular embodiments is notintended to be limiting. As used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to polymer bearing a protected carbonyl would include apolymer bearing two or more protected carbonyls, and the like.

[0056] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, specificexamples of appropriate materials and methods are described herein.

[0057] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0058] The term “bed volume” as used herein is defined as the volume ofa bed of extraction media in an extraction column. Depending on howdensely the bed is packed, the volume of the extraction media in thecolumn bed is typically about half to one third of the total bed volume;well packed beds have less space between the beads and hence generallyhave lower interstital volumes.

[0059] The term “interstitial volume” of the bed refers to the volume ofthe bed of extraction media that is accessible to solvent, e.g, aqueoussample solutions, wash solutions and desorption solvents. For example,in the case where the extraction media is a chromatography bead (e.g.,agarose or sepharose), the interstitial volume of the bed constitutesthe solvent accessible volume between the beads, as well as any solventaccessible internal regions of the bead, e.g, solvent accessible pores.The interstitial volume of the bed represents the minimum volume ofliquid required to saturate the the column bed.

[0060] The term “dead volume” as used herein with respect to a column isdefined as the interstitial volume of the extraction bed, tubes,membrane or frits, and passageways in a column. In the device of thisinvention with gel-type extraction media and the pore volume of thefrits. Since the bottom frit of the column directly contacts the sample,wash, and elution liquids, minimal tubing or passageway dead volume ispresent in this device.

[0061] The term “elution volume” as used herein is defined as the volumeof desorption or elution liquid into which the analytes are desorbed andcollected. The terms “desorption solvent,” elution liquid” and the likeare used interchangeably herein.

[0062] The term “enrichment factor” as used herein is defined as theratio of the sample volume divided by the elution volume, assuming thatthere is no contribution of liquid coming from the dead volume. To theextent that the dead volume either dilutes the analytes or preventscomplete adsorption, the enrichment factor is reduced.

[0063] The terms “extraction column” and “extraction tip” as used hereinare defined as a column device used in combination with a pump, thecolumn device containing a bed of solid phase extraction material, i.e.,extraction media.

[0064] The term “frit” as used herein are defined as porous material forholding the extraction media in place in a column. An extraction mediachamber is typically defined by a top and bottom frit positioned in anextraction column. In preferred embodiments of the invention the frit isa thin, low pore volume filter, e.g, a membrane screen.

[0065] The term “gel-type packing material” as used herein is defined asnon-porous or micro-porous beads such as agarose or sepharose beads, thebeads containing a functional group or having a surface that bindsselectively with the analyte of interest.

[0066] The term “lower column body” as used herein is defined as thecolumn bed and bottom membrane screen of a column.

[0067] The term “membrane screen” as used herein is defined as a wovenor non-woven fabric or screen for holding the column packing in place inthe column bed, the membranes having a low dead volume. The membranesare of sufficient strength to withstand packing and use of the columnbed and of sufficient porosity to allow passage of liquids through thecolumn bed. The membrane is thin enough so that it can be sealed aroundthe perimeter or circumference of the membrane screen so that theliquids flow through the screen.

[0068] The term “sample volume”, as used herein is defined as the volumeof the liquid of the original sample solution from which the analytesare separated or purified.

[0069] The term “upper column body”, as used herein is defined as thechamber and top membrane screen of a column.

[0070] The term “biomolecule” as used herein refers to biomoeculederived from a biological system. The term includes biologicalmacromolecules, such as a proteins, peptides, and nucleic acids.

[0071] The term “protein chip” is defined as a small plate or surfaceupon which an array of separated, discrete protein samples are to bedeposited or have been deposited. These protein samples are typicallysmall and are sometimes referred to as “dots.” In general, a chipbearing an array of discrete proteins is designed to be contacted with asample having one or more biomolecules which may or may not have thecapability of binding to the surface of one or more of the dots, and theoccurrence or absence of such binding on each dot is subsequentlydetermined. A reference that describes the general types and functionsof protein chips is Gavin MacBeath, Nature Genetics Supplement, 32:526(2002).

[0072] II. Low Dead Volume Extraction Columns

[0073] Column Body

[0074] The column body is a tube having two open ends connected by anopen channel. The tube can be in any shape, including but not limited tocylindrical or frustroconical, and of any dimensions consistent with thefunction of the column as described herein. In some preferredembodiments of the invention the column body takes the form of a pipettetip, a syringe, a luer adapter or similar tubular bodies.

[0075] One of the open ends of the column, sometimes referred to hereinas the open upper end of the column, is adapted for attachment to apump. In some embodiments of the invention the upper open end isoperatively attached to a pump, whereby the pump can be used foraspirating a fluid into the extraction column through the other open endof the column, and optionally for discharging fluid out through the openlower end of the column. Thus, it is a feature of the present inventionthat fluid enters and exits the extraction column through the same openend of the column. This is in contradistinction with the operation ofsome extraction columns, where fluid enters the column through one openend and exits through the other end after traveling through anextraction media, i.e, similar to conventional column chromatography.The fluid can be a liquid, such as a sample solution, wash solution ordesorption solvent. The fluid can also be a gas, e.g., air used to blowliquid out of the extraction column.

[0076] The column body can be can be composed of any material that issufficiently non-porous that it can retain fluid and that is compatiblewith the solutions, media, pumps and analytes used. A material should beemployed that does not substantially react with substances it willcontact during use of the extraction column, e.g., the sample solutions,the analyte of interest, the extraction media and desorption solvent. Awide range of suitable materials are available and known to one of skillin the art, and the choice is one of design. Various plastics make idealcolumn body materials, but other materials such as glass, ceramics ormetals could be used in some embodiments of the invention. Some examplesof preferred materials include polysulfone, polypropylene, polyethylene,polyethyleneterephthalate, polyethersulfone, polytetrafluoroethylene,cellulose acetate, cellulose acetate butyrate, acrylonitrile PVCcopolymer, polystyrene, polystyrene/acrylonitrile copolymer,polyvinylidene fluoride, glass, metal, silica, and combinations of theabove listed materials.

[0077] Some specific examples of suitable column bodies are provided inthe Examples.

[0078] Extraction Media

[0079] The extraction media used in the column is preferably a form ofwater-insoluble particle (e.g, a porous or non-porous bead) that has anaffinity for an analyte of interest. Typically the analyte of interestis a protein, peptide or nucleic acid. The extraction processes can beaffinity, reverse phase, normal phase, ion exchange, hydrophobicinteraction chromatography, or hydrophilic interaction chromatographyagents.

[0080] The bed volume of the extraction media used in the extractioncolumns of the invention is typically small, preferably in the range of0.5-100 μL, more preferably in the range of 1-50 μL, and still morepreferably in the range of 2-25 μL. The low bed volume results in a lowinterstitial volume of the bed, contributing to the low dead volume ofthe column, thereby facilitating the recovery of the analyte in a smallvolume of desorption solvent.

[0081] The low bed volumes employed in certain embodiments allow for theuse of relatively small amounts of extraction media, e.g, soft, gel-typebeads. For example, some embodiments of the invention employ a bed ofextraction media having a dry weight of less than 10 mg (e.g., in therange of 0.1-10 mg, 0.5-10 mg, 1-10 mg or 2-10 mg), less than 2 ms(e.g., in the range of 0.1-2 mg, 0.5-2 mg or 1-2 mg), or less than 1 mg(e.g., in the range of 0.1-1 mg or 0.5-1 mg).

[0082] Many of the extraction media types suitable for use in theinvention are selected from a variety of classes of chromatographymedia. It has been found that many of these chromatography media typesand the associated chemistries are suited for use as solid phaseextraction media in the devices methods of this invention.

[0083] Thus, examples of suitable extraction media include agarose-basedmaterials, sepharose-based materials, polystyrene/divinylbenzenecopolymers, poly methylmethacrylate, protein G beads (e.g., for IgGprotein purification), MEP Hypercel™ beads (e.g., for IgG proteinpurification), affinity phase beads (e.g., for protein purification),ion exchange phase beads (e.g., for protein purification), hydrophobicinteraction beads (e.g., for protein purification), reverse phase beads(e.g., for nucleic acid or protein purification), and beads having anaffinity for molecules analyzed by label-free detection. Silica beadsare also suitable.

[0084] Soft gel-type beads, such as agarose and sepharose based beads,are found to work surprisingly well in columns and methods of thisinvention. In conventional chromatography fast flow rates can result inbead compression, which results in increased back pressure and adverselyimpacts the ability to use these gels with faster flow rates. In thepresent invention relatively small bed volumes are used, and it appearsthat this allows for the use of high flow rates with a minimal amount ofbead compression and the problem attendant with such compression.

[0085] Affinity extractions use a technique in which a biospecificadsorbent is prepared by coupling a specific ligand (such as an enzyme,antigen, or hormone) for the analyte, (e.g., macromolecule) of interestto a solid support. This immobilized ligand will interact selectivelywith molecules that can bind to it. Molecules that will not bind eluteunretained. The interaction is selective and reversible. The referenceslisted below show examples of the types of affinity groups that can beemployed in the practice of this invention are hereby incorporated byreference herein in their entireties. Antibody Purification Handbook,Amersham Biosciences, Edition AB, 18-1037-46 (2002); ProteinPurification Handbook, Amersham Biosciences, Edition AC, 18-1132-29(2001); Affinity Chromatography Principles and Methods, AmershamPharmacia Biotech, Edition AC, 18-1022-29 (2001); The RecombinantProtein Handbook, Amersham Pharmacia Biotech, Edition AB, 18-1142-75(2002); and Protein Purification: Principles, High Resolution Methods,and Applications, Jan-Christen Janson (Editor), Lars G. Ryden (Editor),Wiley, John & Sons, Incorporated (1989).

[0086] Examples of suitable affinity binding agents are summarized inTable I, wherein the affinity agents are from one or more of thefollowing interaction categories:

[0087] 1. Chelating metal—ligand interaction

[0088] 2. Protein—Protein interaction

[0089] 3. Organic molecule or moiety—Protein interaction

[0090] 4. Sugar—Protein interaction

[0091] 5. Nucleic acid—Protein interaction

[0092] 6. Nucleic acid—nucleic acid interaction TABLE I Examples ofAffinity molecule or moiety fixed at Interaction surface Capturedbiomolecule Category Ni-NTA His-tagged protein 1 Ni-NTA His-taggedprotein within a 1, 2 multi-protein complex Fe-IDA Phosphopeptides, 1phosphoproteins Fe-IDA Phosphopeptides or 1, 2 phosphoproteins within amulti-protein complex Antibody or other Proteins Protein antigen 2Antibody or other Proteins Small molecule-tagged 3 protein Antibody orother Proteins Small molecule-tagged 2, 3 protein within a multi-protein complex Antibody or other Proteins Protein antigen within a 2multi-protein complex Antibody or other Proteins Epitope-tagged protein2 Antibody or other Proteins Epitope-tagged protein 2 within amulti-protein complex Protein A, Protein G or Antibody 2 Protein LProtein A, Protein G or Antibody 2 Protein L ATP or ATP analogs; 5′-Kinases, phosphatases 3 AMP (proteins that requires ATP for properfunction) ATP or ATP analogs; 5′- Kinase, phosphatases 2, 3 AMP withinmulti-protein complexes Cibacron 3G Albumin 3 Heparin DNA-bindingprotein 4 Heparin DNA-binding proteins 2, 4 within a multi-proteincomplex Lectin Glycopeptide or 4 glycoprotein Lectin Glycopeptide or 2,4 glycoprotein within a multi-protein complex ssDNA or dsDNA DNA-bindingprotein 5 ssDNA or dsDNA DNA-binding protein 2, 5 within a multi-proteincomplex ssDNA Complementary ssDNA 6 ssDNA Complementary RNA 6Streptavidin/Avidin Biotinylated peptides 3 (ICAT) Streptavidin/AvidinBiotinylated engineered tag 3 fused to a protein (see avidity.com)Streptavidin/Avidin Biotinylated protein 3 Streptavidin/AvidinBiotinylated protein within 2, 3 a multi-protein complexStreptavidin/Avidin Biotinylated engineered tag 2, 3 fused to a proteinwithin a multi-protein complex Streptavidin/Avidin Biotinylated nucleicacid 3 Streptavidin/Avidin Biotinylated nucleic acid 2, 3 bound to aprotein or multi- protein complex Streptavidin/Avidin Biotinylatednucleic acid 3, 6 bound to a complementary nucleic acid

[0093] In one aspect of the invention an extraction media is used thatcontains a surface functionality that has an affinity for a proteinfusion tag used for the purification of recombinant proteins. A widevariety of fusion tags and corresponding affinity groups are availableand can be used in the practice of the invention.

[0094] One of the most common fusion tags is the so-called “His” tag,which is comprised of a seris of consecutive histidine residues, e.g,two, four or six consecutive histidine residues. There are a number ofmetal-chelate groups that can be attached to the surface of anextraction media for purification of “His-tagged proteins, includingmetal-IDA (IDA: iminodiacetate), metal-NTA (NTA: nitrilotriacetate), andmetal-CMA (CMA: carboxymethylated aspartate), where the metal istypically selected from nickel, copper, iron, zinc and cobalt. Thetrapped fusion protein is eluted by disrupting the histidine-metalcoordination by some suitable salt such as imidazole or ethylene diaminetetra acetic acid (EDTA).

[0095] There are other affinity groups available for purifyingrecombinant proteins through their fusion tags, and these groups can beattached to an extraction media for use in the invention. Antibodies canbe used for purification through any peptide sequence (a common one isthe FLAG tag); avidin (monomeric or multimeric) can be used forpurifying a peptide sequence that is selectively biotinylated within theexpression system; calmodulin charged with calcium can be used forpurifying a peptide sequence that is often referred to as a “calmodulinbinding peptide” (or, CBP), where elution is performed by removing thecalcium with ethylene glycol tetra acetic acid (EGTA); glutathione canbe used for purifying a fusion protein that carries the glutathioneS-transferase protein (GST), where the GST is often cleaved off with aspecific protease; amylose can be used for purifying a fusion proteinthat carries the maltose binding protein (MBP), where the MBP is oftencleaved off with a specific protease; cellulose can be used forpurifying a fusion protein that carries a peptide that is referred to asthe cellulose-binding domain tag, followed by elution with ethyleneglycol; S-protein (derived from ribonuclease A) can be used forpurifying a fusion protein that carries a peptide with specific affinityfor S-protein, where the peptide can be cleaved off with a specificprotease.

[0096] It is also possible to create an affinity surface that has thebis-arsenical fluorescein dye FIAsH. For example, a FIAsH dye can beused for purifying a fusion protein that carries the peptide sequencetag CCxxCC (where xx is any amino acid, such as RE). The protein is theneluted with 1,4-dithiothreitol, or DTT.

[0097] In one aspect the invention is used for purification ofantibodies. Antibodies are frequently purified on the basis of highlyconserved structural characteristics. For example, it is possible topack columns with extraction media containing Protein A, Protein G, orProtein A/G fusions to purify IgG antibodies through their Fc region(with lower affinity for the Fab antibody fragment region in the case ofProtein G). These are often eluted by using low pH 2.5. It is alsopossible to purify IgG antibodies through their Fab antibody fragmentregion, provided their light chain is a kappa light chain. This isachieved by using a surface of Protein L.

[0098] In one aspect the extraction media comprises small moleculeligands that are capable of achieving separations on the basis ofhydrophobic charge interactions. Ligands such as4-mercapto-ethyl-pyridine and 2-mercaptopyridine are capable of trappingantibodies such as IgGs, which are eluted by changes to low pH muchmilder than in the case of Protein A or Protein G. For example, elutionis accomplished with 4-mercapto-ethyl-pyridine at pH 4 (as opposed to pH2.5 for the Protein A and Protein G).

[0099] In addition, other antibodies can be used for purification ofantibodies. For example, it is possible to use an extraction mediacomprising an immobilized antibody for the purification of IgE (with ananti-IgE surface), the purification of IgM (with an anti-IgM surface),the purification of IgA (with an anti-IgA surface), the purification ofIgD (with an anti-IgD surface), as well as the purification of IgG (withan anti-IgG surface).

[0100] Extraction columns of the invention can be used for purificationof phosphopeptides and phosphoproteins by by the inclusion of anapproprite affinity group on the extraction media. One alternative is toexploit the natural interaction between phosphate groups and metal ions.Therefore, phosphopeptides and phosphoproteins can be purified onmetal-chelate surfaces made from IDA, NTA, or CMA.

[0101] It is also possible to purify these phosphopeptides andphosphoproteins with immobilized antibodies. For example,it is possibleto use antibodies on the packing material that are specific tophosphotyrosine residues, as well as phosphoserine and phosphothreonineresidues. It is also possible to use antibodies that are bind tospecific phophorylated sites within a protein, such asspecifically-binding phosphorylated tyrosine within a specific kinase.These antibodies are often referred to as phosphorylation site-specificantibodies (PSSAs). Once adsorbed the trapped phosphoprotein andphosphopeptides can be eluted at low pH.

[0102] Yet another approach to the purification of phosphopeptides andphosphoproteins involves the derivitization of the phosphate group suchthat biotin is attached to it. This biotinylated phosphoprotein orphosphopeptide can be purified using an avidin-derivatized extractionmedia, wherein the avidin can be monomeric or multimeric.

[0103] In some embodiments of the invention an extraction column is usedfor the purification of protein complexes. One embodiment involves theuse of a recombinant “bait” protein that will form complexes with itsnatural interaction partners. These multiprotein complexes are thenpurified through a fusion tag that is attached to the “bait.” Thesetagged “bait” proteins can be purified through groups incorporated intothe extraction media such as metal-chelate groups, antibodies,calmodulin, or any of the other surface groups described above for thepurification of recombinant proteins.

[0104] It is also possible to purify “native” (i.e. non-recombinant)protein complexes without having to purify through a fusion tag. This isachieved by immobilizing an antibody for one of the proteins within themultiprotein complex. This process is often referred to as“co-immunoprecipitation.” The multiprotein complexes can be eluted withlow pH.

[0105] Extraction columns of the invention can be used to purify entireclasses of proteins on the basis of highly conserved motifs within theirstructure, whereby an affinity ligand attached to the packing reversiblybinds to the conserved motif. For example, it is possible to immobilizeparticular nucleotides on the extraction media. Examples include, butare not limited to, adenosine 5′-triphosphate (ATP), adenosine5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), nicotinamideadenine dinucleotide (NAD), or nicotinamide adenine dinucleotidephosphate (NADP). These nucleotides can be used for the purification ofenzymes that are dependent upon these nucleotides such as kinases,phosphatases, heat shock proteins and dehydrogenases, to name a few.

[0106] There are other affinity groups that can be incorporated into theextraction media for purification of protein classes. Lectins can beused for the purification of glycoproteins. Concanavilin A (Con A) andlentil lectin can be used for the purification of glycoproteins andmembrane proteins, and wheat germ lectin can be used for thepurification of glycoproteins and cells (especially T-cell lymphocytes).Though it is not a lectin, the small molecule phenylboronic acid canalso be incorporated into the extraction media and used for purificationof glycoproteins.

[0107] It is also possible to incorporate heparin intothe extractionmedia, which is useful for the purification of DNA-binding proteins(e.g. RNA polymerase I, II and III, DNA polymerase, DNA ligase). Inaddition, immobilized heparin can be used for purification of variouscoagulation proteins (e.g. antithrombin III, Factor VII, Factor IX,Factor XI, Factor XII and XIIa, thrombin), other plasma proteins (e.g.properdin, BetaIH, Fibronectin, Lipses), lipoproteins (e.g. VLDL, LDL,VLDL apoprotein, HOLP, to name a few), and other proteins (plateletfactor 4, hepatitis B surface antigen, hyaluronidase). These types ofproteins are often blood and/or plasma borne. Since there are manyefforts afoot to rapidly profile the levels of these types of proteinsby technologies such as protein chips, the performance of these chipswill be enhanced by performing an initial purification and enrichment ofthe targets prior to protein chip analysis.

[0108] It is also possible to use extraction media with proteininteraction domains for purification of those proteins that are meant tointeract with that domain. One interaction domain that can be used isthe Src-homology 2 (SH2) domain that binds to specificphophotyrosine-containing peptide motifs within various proteins. TheSH2 domain has previously been immobilized on a resin and used as anaffinity reagent for performing affinity chromatography/massspectrometry experiments for investigating in vitro phosphorylation ofepidermal growth factor receptor (EGFR) (see Christian Lombardo, et al.,Biochemistry, 34:16456 (1995)). Other than the SH2 domain, other proteininteraction domains can be used for the purposes of purifying thoseproteins that possess their recognition domains. Many of these proteininteraction domains have been described (see Tony Pawson, ProteinInteraction Domains, Cell Signaling Technology Catalog, 264-279 (2002))for additional examples of these protein interaction domains).

[0109] Benzamidine is another example of a class-specific affinityligand, which can be incorporated into the extraction media forpurification of serine proteases. The dye ligand Procion Red HE-3B canbe used for the purification of dehydrogenases, reductases andinterferon, to name a few.

[0110] Reversed-phase chromatography media can also function as anextraction media in certain embodiments of the invention. Inreversed-phase chromatography, an aqueous/organic solvent mixture iscommonly used as the mobile phase, and a high-surface-area nonpolarsolid is employed as the stationary phase. The latter can be analkyl-bonded silica packing, e.g., with C₈ or C₁₈ groups covering thesilica surface. The basis of solute retention in reversed-phasechromatography is still somewhat controversial; some workers favor anadsorption, while others believe that the solute partitions into thenonpolar stationary phase. Probably both processes are important formany samples. Competition between solute and mobile-phase moleculesexists for a place on the stationary-phase surface. That is, an adsorbedmolecule will displace some number of previously adsorbed molecules(Chromatography, 5^(th) edition, PART A: FUNDAMENTALS AND TECHNIQUES,editor: E. Heftmann, Elsevier Science Publishing Company, New York, ppA25 (1992)). The near universal application of reversed-phasechromatography stems from the fact that virtually all organic moleculeshave hydrophobic regions in their structure and are capable ofinteracting with the stationary phase. Since the mobile phase is polarand generally contains water, the method is ideally suited to theseparation of polar molecules which are either insoluble in organicsolvents or bind too strongly to inorganic oxide adsorbents for normalelution. Reversed-phase chromatography employing acidic, low ionicstrength eluents has become a widely established technique for thepurification and structural elucidation of proteins. However, thestructure of biopolymers is very sensitive to mobile phase composition,pH and the presence of complexing species which can result in anomalousretention and even denaturing of proteins. A general characteristic ofreversed-phase systems is that a decrease in polarity of the mobilephase, that is increasing the volume fraction of organic solvent in anaqueous organic mobile phase, leads to a decrease in retention; areversal of the general trends observed in liquid-solid chromatographyor normal phase chromatography. It is also generally observed forreversed-phase chromatography that for members of a homologous oroligomous series, the logarithm of the solute capacity factor is alinear function of the number of methylene groups or repeat units of theoligomeric structure (ADVANCED CHROMATOGRAPHIC AND ELECTROMIGRATIONMETHODS IN BIOSCIENCES, editor: Z. Deyl, Elsevier Science BV, Amsterdam,The Netherlands, pp 528 (1998); CHROMATOGRAPHY TODAY, Colin F. Poole andSalwa K. Poole, and Elsevier Science Publishing Company, New York, pp394 (1991)).

[0111] The references listed below show different types of surfaces usedfor reverse phase separations and are hereby incorporated by referenceherein in their entireties: CHROMATOGRAPHY, 5^(th) edition, Part A:Fundamentals and Techniques, editor: E. Heftmann, Elsevier SciencePublishing Company, New York, pp A25 (1992); ADVANCED CHROMATOGRAPHICAND ELECTROMIGRATION METHODS IN BIOSCIENCES, editor: Z. Deyl, ElsevierScience BV, Amsterdam, The Netherlands, pp 528 (1998); CHROMATOGRAPHYTODAY, Colin F. Poole and Salwa K. Poole, and Elsevier SciencePublishing Company, New York, pp 394 (1991).

[0112] Ion-pair chromatography media can also function as an extractionmedia in certain embodiments of the invention. In ion-pairchromatography, the column packing is usually the same as inreversed-phase chromatography; e.g., a C₈ or C₁₈ silica. The mobilephase is likewise similar to that used in reverse phase chromatography:an aqueous/organic solvent mixture containing a buffer plus a so-calledion-pair reagent. The ion-pair reagent will be positively charged forthe retention and separation of sample anions and negatively charged forthe retention of sample cations. Typical examples of ion-pair reagentsare hexane sulfonate and tetrabutylammonium. The basis of retention inion-pair chromatography is still controversial, two different processesbeing possible: (a) adsorption of ion pairs or (b) formation of an insitu ion exchanger. Although these two processes appear somewhatdifferent, they lead to quite similar predictions of retention as afunction of experimental conditions. Retention in ion-pairchromatography can be continuously varied from a reversed-phase processto an ion-exchange process. This capability provides a number ofpractical advantages. For example, variation of the mobile phasecomposition allows a considerable control over the retention ofindividual sample ions. This can be used to separate particularlydifficult samples, e.g., mixtures of anionic, cationic, and/or neutralmolecules (CHROMATOGRAPHY, 5^(th) Edition, Part A: Fundamentals AndTechniques, editor: E. Heftmann, Elsevier Science Publishing Company,New York, pp A28 (1992)).

[0113] The references listed below show different types of groups usedfor ion-pair chromatography and are hereby incorporated by referenceherein in their entireties: Reference: CHROMATOGRAPHY, 5^(th) Edition,Part A: Fundamentals and Techniques, editor: E. Heftmann, ElsevierScience Publishing Company, New York, pp A28 (1992); and CHROMATOGRAPHYTODAY, Colin F. Poole and Salwa K. Poole, Elsevier Science PublishingCompany, New York, pp 411 (1991).

[0114] Normal phase chromatography media can also function as anextraction media in certain embodiments of the invention. In normalphase chromotagraphy, the stationary phase is a high-surface-area polaradsorbent, e.g., silica or a bonded silica with polar surface groups.The mobile phase (a mixture of organic solvents) is less polar than thestationary phase. Consequently, more polar solutes are preferentiallyretained; there is often little difference in the retention of differenthomologs or a particular compound class. This has led to the use ofnormal phase chromatography for so-called compound-class (group-type)separations, where, e.g., alcohols are separated as a group frommonoesters and other compound classes. The basis of normal phasechromatography retention is an adsorption/displacement process. Anotherfeature of normal phase chromatography retention is the so-calledlocalization of adsorbed solute and mobile-phase molecules on thestationary-phase surface. Localization refers to the formation ofdiscrete bonds (by dipole/dipole or hydrogen-bonding interactions)between polar sites on the adsorbent and polar substituents in thesolute molecule. Localization, in turn, confers a high degree ofspecificity to the interaction of solute isomers with the adsorbentsurface, leading to typically better separations of isomers by normalphase chromatography than by other chromatographic methods(CHROAMTOGRAPHY, 5^(th) edition, Part A: Fundamentals and Techniques,editor: E. Heftmann, Elsevier Science Publishing Company, New York, ppA27 (1992)).

[0115] The references listed below show different types of affinitygroups used for normal phase chromatography and are hereby incorporatedby reference herein in their entireties: CHROMATOGRAPHy, 5^(th) edition,Part A: Fundamentals and Techniques, editor: E. Heftmann, ElsevierScience Publishing Company, New York, pp A27 (1992); and CHROMATOGRAPHYTODAY, Colin F. Poole and Salwa K. Poole, Elsevier Science PublishingCompany, New York, pp 375 (1991).

[0116] Ion Exchange chromatography media can also function as anextraction media in certain embodiments of the invention. Ion Exchange(IEX) is a mode of chromatography in which ionic substances areseparated on cationic or anionic sites of the packing. The surface inion exchange is usually an organic matrix which is substituted withionic groups, e.g., sulfonate or trimethylammonium. The mobile phasetypically consists of water plus buffer and/or salt. The retention of asolute ion occurs via ion exchange with a mobile phase ion or similar(positive or negative) charge. Ion exchange chromatography is oftenapplied to the separation of acidic or basic samples, whose chargevaries with pH. In the simple case of solute molecules bearing a singleacidic or basic group, the solute will be present as some mixture ofcharged and neutral species. The fraction of solute molecules that areionized then determines retention. In the case of ion exchange, theretention of the uncharged species can be ignored (CHROMATOGRAPHY,5^(th) Edition, Part A: Fundamentals and Techniques, editor: E.Heftmann, Elsevier Science Publishing Company, New York, pp A28 (1992)).Ion exchange chromatography is one of the oldest and most traditionaltechniques for separating complex mixtures of proteins. The referenceslisted below show different types of groups and surfaces used for ionexchange chromatography and are hereby incorporated by reference hereinin their entireties; CHROMATOGRAPHY, 5^(th) Edition, Part A:Fundamentals and Techniques, editor: E. Heftmann, Elsevier SciencePublishing Company, New York, pp A28 (1992); CHROMATOGRAPHY TODAY, ColinF. Poole and Salwa K. Poole, Elsevier Science Publishing Company, NewYork, pp 422 (1991); and ADVANCED CHROMATOGRAPHIC AND ELECTROMIGRATIONMETHODS IN BIOSCIENCES, editor: Z. Deyl, Elsevier Science BV, Amsterdam,The Netherlands, pp 540 (1998).

[0117] Hydrophobic Interaction Chromatography media can also function asan extraction media in certain embodiments of the invention. HydrophobicInteraction Chromatography is widely used for the separation andpurification of proteins. During separation, proteins are induced tobind to a weakly hydrophobic stationary phase using a buffered mobilephase of high ionic strength and then selectively desorbed during adecreasing salt concentration gradient. Proteins are usually separatedin hydrophobic interaction chromatography according to their degree ofhydrophobicity, much as in reversed-phase chromatography, but because ofthe gentler nature of the separation mechanism, there is a greaterprobability that they will elute with their conformational structure(biological activity) intact. In reversed-phase chromatography, proteinsunfold on the bonded phase surface as a consequence of the highinterfacial tension existing between the mobile and the bondedstationary phases. These conditions are minimized in hydrophobicinteraction chromatography by using stationary phases of lowerhydrophobicity together with totally aqueous mobile phases, in general,since solvent strength is controlled by varying ionic strength ratherthan by increasing the volume fraction of an organic modifier. Retentionand selectivity in hydrophobic interaction chromatography dependsubstantially on the type of stationary phase. Retention increases formore hydrophobic ligands and with it the possibility of denaturingcertain proteins. Some proteins are only satisfactorily handled onhydrophilic stationary phases. The ligand density and structure as wellas the hydrophobicity of the stationary phase are the primary stationaryphase variables that should be optimized for the separation ofindividual proteins. Mobile phase parameters that have to be optimizedare the salt concentration, salt type, slope of the salt gradient, pH,addition of surfactant or organic modifier and temperature. In theabsence of specific binding of the salt to the protein molecule and atrelatively high salt concentration in the mobile phase, retentionincreases linearly with the salt molality and at constant saltconcentration with the molal surface tension increment of the salt usedin the aqueous mobile phase.

[0118] The reference listed below shows different types of groups andsurfaces used for hydrophobic interactions and is hereby incorporated byreference herein in its entirety: CHROMATOGRAPHY TODAy, Colin F. Pooleand Salwa K. Poole, Elsevier Science Publishing Company, New York, 402(1991).

[0119] Frits

[0120] Columns of the invention employ frits having a low pore volume,which contributes to the low dead volume of the columns. The frits ofthe invention are porous, since it is necessary for fluid to be able topass through the frit. The frit should have sufficient structuralstrength so that frit integrity can contain the extraction media in thecolumn. It is desirable that the frit have little or no affinity forchemicals with which it will come into contact during the extractionprocess, particularly the analyte of interest. In many embodiments ofthe invention the analyte of interest is a biomolecule, particularly abiological macromolecule. Thus in many embodiments of the invention itdesirable to use a frit that has a minimal tendency to bind or otherwiseinteract with biological macromolecules, particularly proteins, peptidesand nucleic acids.

[0121] Frits of various pores sizes and pore densities may be usedprovided the free flow of liquid is possible and the beads are held inplace within the extraction media bed.

[0122] One frit, i.e., a lower frit, is bonded to and extends across theopen channel of the column body. A second frit is bonded to and extendsacross the open channel between the bottom frit and the open upper endof the column body.

[0123] The top frit, bottom frit and channel surface define anextraction media chamber wherein a bed of extraction media ispositioned. The frits should be securely attached to the column body andextend across the opening and/or open end so as to completely occludethe channel, thereby substantially confining the bed of extraction mediainside the extraction media chamber. In preferred embodiments of theinvention the bed of extraction media occupies at least 80% of thevolume of the extraction media chamber, more preferably 90%, 95%, 99%,or substantially 100% of the volume. In some preferred embodiments theinvention the space between the extraction media bed and the upper andlower frits is negligible, i.e., the frits are in substantial contactwith upper and lower surfaces of the extraction media bed, holding awell-packed bed of extraction media securely in place.

[0124] In some preferred embodiments of the invention the bottom frit islocated at the open lower end of the column body. This configuration isshown in the figures and exemplified in the Examples, but is notrequired, i.e., in some embodiments the bottom frit is located at somedistance up the column body from the open lower end. However, in view ofthe importance of minimizing dead volume in the column it is desirablethat the lower frit and extraction media chamber be located at or nearthe lower end. In some cases this can mean that the bottom frit isattached to the face of the open lower end, as shown in FIGS. 1-10.However, in some cases there can be some portion of the lower endextending beyond the bottom frit, as exemplified by the embodimentdepicted in FIG. 11. For the purposes of this invention, so long as thelength as this extension is such that it does not substantiallyintroduce dead volume into the extraction column or otherwise adverselyimpact the function of the column, the bottom frit is considered to belocated at the lower end of the column body. In some embodiments of theinvention the volume defined by the bottom frit, channel surface, andthe face of the open lower end (i.e., the channel volume below thebottom frit) is less than the volume of the extraction media chamber, orless than 10% of the volume of the extraction media chamber, or lessthan 1% of the volume of the extraction media chamber.

[0125] The frits used in the invention are characterized by having a lowpore volume. Some preferred embodiments invention employ frits havingpore volumes of less than 1 microliter (e.g., in the range of 0.015-1microliter, 0.03-1 microliter, 0.1-1 microliter or 0.5-1 microliter),preferably less than 0.5 microliter (e.g., in the range of 0.015-0.5microliter, 0.03-0.5 microliter or 0.1-0.5 microliter), less than 0.1microliter (e.g., in the range of 0.015-0.1 microliter or 0.03-0.1microliter) or less than 0.03 microliters (e.g., in the range of0.015-0.03 microliter).

[0126] Frits of the invention preferably have pore openings or meshopenings of a size in the range of about 5-100 μm, more preferably10-100 μm, and still more preferably 15-50 μm, e.g, about 43 μm. Theperformance of the column is typically enhanced by the use of fritshaving pore or mesh openings sufficiently large so as to minimize theresistance to flow. The use of membrane screens as described hereintypically provide this low resistance to flow and hence better flowrates, reduced back pressure and minimal distortion of the bed ofextraction media. The pre or mesh openings of course should not be solarge that they are unable to adequately contain the extraction media inthe chamber.

[0127] The frits used in the practice of the invention are characterizedby having a low pore volume relative to the interstitial volume of thebed of extraction media contained by the frit. Thus, in preferredembodiments of the invention the frit pore volume is equal to 10% orless of the interstitial volume of the bed of extraction media (e.g., inthe range 0.1-10%, 0.25-10%, 1-10% or 5-10% of the interstitial volume),more preferably 5% or less of the interstitial volume of the bed ofextraction media (e.g., in the range 0.1-5%, 0.25-5% or 1-5% of theinterstitial volume), and still more preferably 1% or less of theinterstitial volume of the bed of extraction media (e.g., in the range0.1-1% or 0.25-0% of the interstitial volume).

[0128] The pore density will allow flow of the liquid through themembrane and is preferably 10% and higher to increase the flow rate thatis possible and to reduce the time needed to process the sample.

[0129] Some embodiments of the invention employ a thin frit, preferablyless than 350 μm in thickness (e.g., in the range of 20-350 μm, 40-350μm, or 50-350 μm), more preferably less than 200 μm in thickness (e.g.,in the range of 20-200 μm, 40-200 μm, or 50-200 μm), more preferablyless than 100 μm in thickness (e.g., in the range of 20-100 μm, 40-100μm, or 50-100 μm), and most preferably less than 75 μm in thickness(e.g., in the range of 20-75 μm, 40-75 μm, or 50-75 μm).

[0130] Some preferred embodiments of the invention employ a membranescreen as the frit. The membrane screen should be strong enough to notonly contain the extraction media in the column bed, but also to avoidbecoming detached or punctured during the actual packing of the mediainto the column bed. Membranes can be fragile, and in some embodimentsmust be contained in a framework to maintain their integrity during use.However, it is desirable to use a membrane of sufficient strength suchthat it can be used without reliance on such a framework. The membranescreen should also be flexible so that it can conform to the column bed.This flexibility is advantageous ins the packing process as it allowsthe membrane screen to conform to the bed of extraction media, resultingin a reduction in dead volume.

[0131] Preferably the membrane is a woven or non-woven mesh of fibersthat may be a mesh weave, a random orientated mat of fibers i.e. a “”polymer paper,” a spun bonded mesh, an etched or “pore drilled” paperor membrane such as nuclear track etched membrane or an electrolyticmesh (see, e.g., 5,556,598). The membrane may be polymer, glass, ormetal provided the membrane is low dead volume, allows movement of thevarious sample and processing liquids through the column bed, may beattached to the column body, is strong enough to withstand the bedpacking process, is strong enough to hold the column bed of beads, anddoes not interfere with the extraction process i.e. does not adsorb ordenature the sample molecules.

[0132] The frit can be attached to the column body by any means whichresults in a stable attachment. For example, the screen can be bonded tothe column body through welding or gluing. Gluing can be done with anysuitable glue, e.g., silicone, cyanoacrylate glue, epoxy glue, and thelike. The glue or weld joint must have the strength required towithstand the process of packing the bed of extraction media and tocontain the extraction media with the chamber. For glue joints, a glueshould be selected employed that does not adsorb or denature the samplemolecules.

[0133] Alternatively, a frit can be attached by means of an annular pip,as described in U.S. Pat. No. 5,833,927. This mode of attachment isparticularly suited to embodiment where the frit is a membrane screen.

[0134] The frits of the invention, e.g., a membrane screen, can be madefrom any material that has the required physical properties as describedherein. Examples of suitable materials include nylon, polyester,polyamide, polycarbonate, cellulose, polyethylene, nitrocellulose,cellulose acetate, polyvinylidine difluoride, polytetrafluoroethylene(PTFE), polypropylene and glass. A specific example of a membrane screenis the 43 μm pore size Spectra/Mesh® polyester mesh material which isavailable from Spectrum Labs (Ranch Dominguez, Calif., PN 145837).

[0135] Extraction Column Assembly

[0136] The extraction columns of the invention can be constructed by avariety of methods using the teaching supplied herein. In some preferredembodiments the extraction column can be constructed by the engagement(i.e., attachment) of upper and lower tubular members that combine toform the extraction column. Examples of this mode of column constructionare described in the Examples and depicted in the figures.

[0137] For example, an embodiment of the invention wherein in the twotubular members are sections of pipette tips is depicted in FIG. 1 (FIG.2 is an enlarged view of the open lower end and extraction media chamberof the column). This embodiment is constructed from a frustoconicalupper tubular member 2 and a frustoconical lower tubular member 3engaged therewith. The engaging end 4 of the lower tubular member has atapered bore that matches the tapered external surfaced of the engagingend 6 of the upper tubular member, the engaging end of the lower tubularmember receiving the engaging end of the upper tubular member in atelescoping relation. The tapered bore engages the tapered externalsurface snugly so as to form a good seal in the assembled column.

[0138] A membrane screen 10 is bonded to and extends across the tip ofthe engaging end of the upper tubular member and constitutes the upperfrit of the extraction column. Another membrane screen 14 is bonded toand extends across the tip of the lower tubular member and constitutesthe lower frit of the extraction column. The extraction media chamber 16is defined by the membrane screens 10 and 14 and the channel surface 18,and is packed with extraction media 20.

[0139] The pore volume of the membrane screens 10 and 14 is low tominimize the dead volume of the column. The sample and desorptionsolution can pass directly from the vial or reservoir into the bed ofextraction media. The low dead volume permits desorption of the analyteinto the smallest possible desorption volume, thereby maximizing analyteconcentration.

[0140] The volume of the extraction media chamber 16 is variable and canbe adjusted by changing the depth to which the upper tubular memberengaging end extends into the lower tubular member, as determined by therelative dimensions of the tapered bore and tapered external surface.

[0141] The sealing of the upper tubular member to the lower tubular inthis embodiment is achieved by the friction of a press fit, but couldalternatively be achieved by welding, gluing or similar sealing methods.

[0142]FIG. 3 depicts an embodiment of the invention comprising an upperand lower tubular member engaged in a telescoping relation that does notrely on a tapered fit. Instead, in this embodiment the engaging ends 34and 35 are cyclindrical, with the outside diameter of 34 matching theinside diameter of 35, so that he concentric engaging end form a snugfit. The engaging ends are sealed through a press fit, welding, gluingor similar sealing methods. The volume of the extraction bed can bevaried by changing how far the length of the engaging end 34 extendsinto engaging end 35. Note that the diameter of the upper tubular member30 is variable, in this case it is wider at the upper open end 31 andtapers down to the narrower engaging end 34. This design allows for alarger volume in the column channel above the extraction media, therebyfacilitating the processing of larger sample volumes and wash volumes.The size and shape of the upper open end can be adapted to conform to apump used in connection with the column. For example, upper open end 31can be tapered outward to form a better friction fit with a pump such asa pipettor or syringe.

[0143] A membrane screen 40 is bonded to and extends across the tip 38of engaging end 34 and constitutes the upper frit of the extractioncolumn. Another membrane screen 44 is bonded to and extends across thetip 42 of the lower tubular member 36 and constitutes the lower frit ofthe extraction column. The extraction media chamber 46 is defined by themembrane screens 40 and 44 and the open interior channel of lowertubular member 36, and is packed with extraction media 48.

[0144]FIG. 4 is a syringe pump embodiment of the invention with acyclindrical bed of extraction media in the tip, and FIG. 5 is anenlargement of the top of the syringe pump embodiment of FIG. 4. Thesefigures show a low dead volume column based on using a disposablesyringe and column body. Instead of a pipettor, a disposable syringe isused to pump and contain the sample.

[0145] The upper portion of this embodiment constitutes a syringe pumpwith a barrel 50 into which a plunger 52 is positioned for movementalong the central axis of the barrel. A manual actuator tab 54 issecured to the top of the plunger 52. A concentric sealing ring 56 issecured to the lower end of the plunger 52. The outer surface 58 of theconcentric sealing ring 56 forms a sealing engagement with the innersurface 60 of the barrel 50 so that movement of the plunger 52 andsealing ring 56 up or down in the barrel moves liquid up or down thebarrel.

[0146] The lower end of the barrel 50 is connected to an inner cylinder62 having a projection 64 for engaging a Luer adapter. The bottom edge66 of the inner cylinder 62 has a membrane screen 68 secured thereto.The inner cylinder 62 slides in an outer sleeve 70 with a conventionalLuer adaptor 72 at its upper end. The lower segment 74 of the outersleeve 70 has a diameter smaller than the upper portion 76, outer sleeve70 forming a ledge 78 positioned for abutment with the lower end 66 andmembrane screen 68. A membrane screen 80 is secured to the lower end 82of the lower segment 74. The extraction media chamber 84 is defined bythe upper and lower membrane screens 68 and 80 and the inner channelsurface of the lower segment 74. The extraction beads 86 are positionedin the extraction media chamber 84. The volume of extraction mediachamber 84 can be adjusted by changing the length of the lower segment74.

[0147] Other embodiments of the invention exemplifying different methodsof construction are also described in the examples.

[0148] Pump

[0149] In using the extraction columns of the invention a pump isattached to the upper open end of the column and used to aspirated anddischarge the sample from the column. The pump can take any of a varietyof forms, so long as it is capable of generating a negative internalcolumn pressure to aspirate a fluid into the column channel through theopen lower end. In some preferred embodiments of the invention the pumpis also able to generate a positive internal column pressure todischarge fluid out of the open lower end. Alternatively, other methodscan be used for discharging solution from the column, e.g,centrifugation.

[0150] The pump should be capable of pumping liquid or gas, and shouldbe sufficiently strong so as to be able to draw a desired samplesolution, wash solution and/or desorption solvent through the bed ofextraction media.

[0151] In some preferred embodiments of the invention the pump iscapable of controlling the volume of fluid aspirated and/or dischargedfrom the column, e.g, a pipettor. This allows for the metered intake andouttake of solvents, which facilitates more precise elution volumes tomaximize sample recovery and concentration.

[0152] Non-limiting examples of suitable pumps include a pipettor,syringe, peristaltic pump, electrokinetic pump, or an induction basedfluidics pump.

[0153] III. Methods for Using the Extraction Columns

[0154] Extraction columns of the invention should be stored underconditions that preserve the integrity of the extraction media. Forexample, columns containing agarose- or sepharose-based extraction mediashould be stored under cold conditions (e.g., 4 degrees Celsius) and inthe presence of 0.01 percent sodium azide or 20 percent ethanol.

[0155] The sample solution can be any solution containing an analyte ofinterest. The invention is particularly useful for extraction andpurification of biological molecules, hence the sample solution is oftenof biological origin, e.g., a cell lysate. In one embodiment of theinvention the sample solution is a hybridoma cell culture supernatant.

[0156] Prior to extraction, a conditioning step may be employed. Ifanalyte extraction is incomplete in a single pass, the sample solutioncan be passed back and forth through the media several times. Anoptional wash step between the extraction and desorption steps can alsoimprove the purity of the final product. Typically water or a buffer isused for the wash solution. The wash solution is preferably one thatwill remove unwanted contaminants with a minimal desorption of theanalyte of interest.

[0157] The volume of desorption solvent used can be very small,approximating the interstitial volume of the bed of extraction media. Inpreferred embodiments of the invention the amount of desorption solventused is less than 10-fold greater than the interstitial volume of thebed of extraction media, more preferably less than 5-fold greater thanthe interstitial volume of the bed of extraction media, still morepreferably less than 3-fold greater than the interstitial volume of thebed of extraction media, still more preferably less than 2-fold greaterthan the interstitial volume of the bed of extraction media, and mostpreferably is equal to or less than the interstitial volume of the bedof extraction media.

[0158] The desorption solvent will vary depending upon the nature of theanalyte and extraction media. For example, where the analyte is ahis-tagged protein and the extraction media an IMAC resin, thedesorption solution will contain imidazole or the like to release theprotein from the resin. In some cases desorption is achieved by a changein pH or ionic strength, e.g., by using low pH or high ionic strengthdesorption solution. A suitable desorption solution can be arrived atusing available knowledge by one of skill in the art.

[0159] In one embodiment, the extraction column may be used formultidimensional stepwise solid phase extraction of isotope-codedaffinity tagged (ICAT) peptides. The fractions are collected on thebasis of increasing ionic strength or pH, and can be processed in theaffinity separation dimension described below, but with suitableadjustments being made for larger sample volumes being introduced intothe affinity capillary and/or possible differences in pH. In certaininstances the fractions collected from the avidin affinity column may beprocessed further for cleavage of the affinity tag from theisotope-coding region, prior to separation in the reversed-phaseseparation dimension described below.

[0160] The cleavage can be performed directly upon the collectedfraction by photocleavage as described in Huilin Zhou, et al., NatureBiotech., 19:512 (2002), or acid cleavage with TFA-triethylsilane asdescribed in Brian Williamson, et al., Proceedings of the 50^(th) ASMSConference on Mass Spectrometry and Allied Topics, Orlando, Fla., Jun.2-6, 2002, Orlando, Fla., Poster # WPA023, or by evaporating thecollected fraction to dryness by standard means and addingTFA-triethylsilane reagent to achieve acid cleavage as described inWilliamson, et al, 50^(th) ASMS Conference Proceedings, Jun.2^(nd)-6^(th) 2002, Orlando, Fla., Poster # WPA023 (2002).

[0161] In instances where the peptide mixture generated by the release,labeling and proteolysis is not excessively complex, it may be possibleto bypass the ion-exchange separation dimension and proceed directly tothe affinity separation dimension. An example of bypassing theion-exchange separation dimension is given in LC Packings/Dionex'Application Note, “2D Analysis of Isotope Coded Affinity Tag (ICAT)Labeled Proteins,” Application Note UltiMate Capillary and Nano LCSystem, Proteomics #09. However, if this strategy is applied it isadvised that some suitable means be applied for removal of theunincorporated ICAT tags prior to introducing the sample to themonomeric avidin column, which would otherwise be removed in theion-exchange separation dimension.

[0162] In certain instances it may be possible to bypass theion-exchange separation and affinity separation dimensions and proceeddirectly from the sample protein release, lysis and labeling step (i.e.the first step described at the beginning of this example) to thereversed-phase separation dimension, such as when solid-phaseisotope-coded tagging reagents are being utilized as described in HuilinZhou, et al., Nature Biotech., 19:512 (2002); in this case the cleavageof the isotope-coded peptide from the solid-phase support can beachieved by photocleavage as described in Huilin Zhou, et al., NatureBiotech., 19:512 (2002) or by acid cleavage as described in BrianWilliamson, et al., Proceedings of the 50^(th) ASMS Conference on MassSpectrometry and Allied Topics, Orlando, Fla., Jun. 2-6, 2002, Orlando,Fla., Poster # WPA023.

[0163] The device, apparatus and method of this invention can be used toprepare materials for protein chips, DNA chips or other biochips.

[0164] Protein chips dynamics can be represented by the followingequation:

A+B=AB

[0165] AB is capable of generating an analytical signal, where A is thechip-bound moiety and B is its cognate binder introduced to the chip. Anassumption of specific interactions is always assumed. Binding eventsother than “AB” can have the appearance of AB, the variance being causedby non-A (i.e. contaminating) moieties having some affinity for B, non-B(i.e. contaminating) moieties having some affinity for A, or acombinations of the two; any of these events will have the appearance ofa true AB event. This characteristic will define the success or failureof a particular protein chip experiment, and is the most trivialized orignored aspects of the technology.

[0166] For some non-protein chips (specifically DNA chips), the A groupsdo not require purification or enrichment since they are synthesized inplace, or are amplified via PCR and spotted. With the exception of veryshort peptides, the structural complexity of proteins will not allow foron-chip synthesis of A. Therefore, preparation of A materials for usewithin protein chips will place a premium on the purity of the material.In addition, the A materials will often need to be highly enriched so asto provide maximum opportunity for AB to occur.

[0167] Protein chips are characterized by having small volumes of “A”applied to the surface. The volumes are often on the order of 10 nL orless for each spot. Since many proteins are difficult and/or expensiveto prepare, the ability to purify and enrich at scales on par with thespots would significantly reduce waste. It would also allow for“just-in-time” purification, so that the chip is prepared just as theprotein is being purified.

[0168] Different materials are brought to the chip as A, and eachmaterial require purification and/or enrichment. Examples of thesematerials are antibodies (i.e. IgG, IgY, etc) as affinity molecules,general affinity proteins (i.e. scFvs, Fabs, affibodies, peptides, etc)as affinity molecules, other proteins that are being screened forgeneral affinity characteristics, and nucleic acids/(photo) aptamers asaffinity molecules, for example.

[0169] Different means of attaching A to chip surfaces, and each willrequire purification and enrichment procedures that are compatible withthe attachment chemistry. Examples of attachment chemistry includedirect/passive immobilization to protein chip substrates, and these canbecome covalent in cases of native thiols associating with goldsurfaces, as one example. Covalent attachment is another method ofattachment of functional groups at chip surface, and these can beself-assembled monolayers with and without additional groups,immobilized hydrogel, and the like. Non-covalent/affinity attachment tofunctional groups/ligands at chip surface is another method ofattachment; examples of this method are ProA or ProG for IgGs,phenyl(di)boronic acid with salicylhydroxamic acid groups; streptavidinmonolayers with biotinylation of native lysines/cysteines, and the like.

[0170] The samples or analyte to be brought to the chip can be varied incomposition and mode of interaction with A.

[0171] There is more than one way to achieve specific AB interactionsthrough the manipulation of B. One means is to remove potentiallyinterfering non-B contaminants by their specific removal, provided thesecontaminants are sufficiently well-defined such as albumin, fibrin, etc.

[0172] Another means is the removal of non-B contaminants by trapping B(either individually or as a class), removing contaminants by washing,and releasing B. This simultaneously allows for enrichment of B, thusenhancing the sensitivity for the AB event.

[0173] Just as the scale of the chip is very small, there areopportunities to make the scale of the sample small—therefore allowingfor analysis of very small samples. Since samples are preciousmaterials, the scale of purification and enrichment would allow for thisto occur. As with chip preparation, this can occur in a “just-in-time”manner.

[0174] The detection event requires some manner of A interacting with B,so the central player in the detection event (since it isn't part of theprotein chip itself) is B. The means of detecting the presence of B (or,B-like substances described above) are varied and can include label-freedetection of B (or B-like substances) interacting with A such as surfaceplasmon resonance imaging as practiced by HTS Biosystems—grating-coupledSPR or BiaCore—prism or Kretschmann-based SPR, or Micro-cantileverdetection schemes as practiced by Protiveris.

[0175] The detection means can include physical labeling of B (or B-likesubstances) interacting with A, followed by spatial imaging of AB pair(i.e. Cy3/Cy5 differential labeling with standard fluorescent imaging aspracticed by BD Biosciences Clontech, radioactive ATP labeling of kinasesubstrates with autoradiography imaging as practiced by Jerini or othersuitable imaging techniques. In the case of fluorescent tagging, one canachieve higher sensitivity with fluorescent waveguide imaging aspracticed by ZeptoSens.

[0176] The detection means can also include interaction of AB complexwith a third B-specific affinity partner C, where C is capable ofgenerating a signal by being fluorescently tagged, or is tagged with agroup that allows a chemical reaction to occur at that location (such asgeneration of a fluorescent moiety, direct generation of light, etc).Detection of this AB-C binding event can occur via fluorescent imagingas practiced by Zyomyx and SomaLogic, chemilumine-scence imaging aspracticed by HTS Biosystems and Hypromatrix, fluorescent imaging viawaveguide technology, or other suitable detection means.

[0177] Arrayers are instruments for spotting nucleic acids, proteins orother reagent onto chips that are used for molecular biology research ordiagnostic work. The arrayers can be used both in the manufacture of thechips and in the use of the chip. In manufacturing, an arrayer can beused to transport the chemical reactants to specific spots on the chip.This may be a multistep process as the chemical complex used fordetection is built at each particular spot in the array.

[0178] Each process can require sample preparation. In some cases, DNAis purified and deposited to a surface on a chip. Then samplescontaining complementary DNA or RNA are reacted with the chip. Beforethe samples can be reacted, the nucleic acid is purified away from theother materials (proteins, particulate, carbohydrates, etc.) found inthe samples. In other cases, protein chips may be manufactured bydepositing specific proteins in an array. Then samples containingproteins can be reacted with various array sites to measureprotein/protein interactions.

[0179] In application of mass spectrometry for the analysis ofbiomolecules, the molecules must be transferred from the liquid or solidphases to gas phase and to vacuum phase. Since most biomolecules areboth large and fragile, the most effective methods for their transfer tothe vacuum phase are matrix-assisted laser desorption ionization (MALDI)or electrospray ionization (ESI).

[0180] Mass spectrometry provides essentially two methods for analyzingproteins: bottom up and top down analysis. In bottom up analysis, theprotein is manipulated and broken up in a controlled manner (usuallythrough an enzymatic digestion process), analyzed, and then reassembledusing the data from the various parts. Top down analysis works with thewhole protein, optionally using an ion source to break apart the proteinand determine the identity of the protein.

[0181] While both methods may require long mass spectrometer analysistimes, top down approaches usually require the longest time. Under idealcases, a static sample is measured and parameters on the manner in whichthe source is directed or implemented. The methods in which the data areanalyzed are varied to perform a full analysis of the protein.

[0182] Many sample introduction methods introduce samples “on-the-fly.”The sample is introduced from an HPLC column as continuous flow into thenozzle of the electrospray ionization (ESI) source. In order tointroduce samples so that top down analysis can be implemented, the flowof the sample may be slowed. The method is called peak parking. In thisway, the sample residence time can be increased by a factor of 10 orgreater increasing the sensitivity of the analysis by a factor of 8 orgreater. However, this method is still inflexible and inadequate becausethe analysis must still be performed quickly—often more quickly than theinstrument is capable of performing.

[0183] This is also true for introduction of samples from a solid phaseextraction device. One may introduce the entire sample before theanalysis is completed. It is much better to introduce a discrete uniformsample into the mass spectrometer. In this way, the mass spectrometrymethod and procedure can be adapted to the sample in the best manner.

[0184] This can be accomplished by using an apparatus where the desorbedmaterial from an open tube extraction device is deposited directly intoan electrospray nozzle.

[0185] MALDI is commonly interfaced to time of flight (TOF) massspectrometers (MALDI-TOF) and ESI is interfaced to quadrupole, ion trapand TOF mass analyzers. Both MALDI and ESI approaches are useful fordetermining the full masses of proteins and peptides in mixtures, beforeand after purification and to induce fragmentation of peptides for ms/msanalysis. Modem mass spectrometry is accurate enough to be useful forevaluating the correct translation or chemical synthesis ofbiomolecules. Any deviation of the observed mass of the sample from itscalculated mass indicates incorrect synthesis or the presence ofpost-translational or chemical modifications. Biomolecules can bepurposely fragmented in the mass spectrometer and the masses of theresulting fragments can be accurately determined. The patterns of suchfragment masses are useful for ms/ms sequencing of the peptides andtheir identification in the data banks.

[0186] Electrospray is performed by mixing the sample with volatile acidand organic solvent and infusing it through a conductive needle chargedwith high voltage. The charged droplets that are sprayed (or ejected)from the needle end, are directed into the mass spectrometer, and aredried up by heat and vacuum as they fly in. After the drops dry, theremaining charged molecules are directed by electromagnetic lenses intothe mass detector and mass analyzed. Electrospray mass spectrometry canbe used to determine the masses of different molecules, from smallpeptides to intact large proteins. Even though the mass-range of thecurrently available instruments is only 2000 to 10000 mass unit, mostproteins become multi-charged during the electrospray step and since theinstrument measures the mass to charge ratio (m/z) of the molecules,most proteins are sufficiently charged to have an m/z that is within themass range. To calculate the full mass of the protein from the differentm/z measured, a deconvolution is performed, returning the full mass ofthe proteins.

[0187] For MALDI-TOF the proteins are deposited on metal targets, asco-crystallized with an organic matrix. The samples are dried andinserted into the mass spectrometer. After vacuum is established, thematrix crystals absorb the light energy from short flashes of ahigh-energy laser. The matrix rapidly sublimes, carrying with it thebiomolecule into the vacuum phase. The sample and matrix plume enter astrong electromagnetic field that accelerate the charged molecules intoa free flight zone where they fly until they hit a detector located atits far end. The mass of the protein can be calculated from its flighttime. Accurate determination of the masses is obtained by the flighttime to that of a standard of known mass. The flight time isproportional to the log of mass of the protein and the larger proteinsfly slower and reach the detector later.

[0188] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0189] Having now generally described the invention, the same will bemore readily understood through reference to the following examples,which are provided by way of illustration, and are not intended to belimiting of the present invention, unless so specified.

EXAMPLES

[0190] The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and practice the presentinvention. They should not be construed as limiting the scope of theinvention, but merely as being illustrative and representative thereof.

Example 1

[0191] Preparation of an Extraction Column Body from Pipette Tips

[0192] Two 1000 μL polypropylene pipette tips of the design shown inFIG. 6 (VWR, Brisbane, Calif., PN 53508-987) were used to construct oneextraction column. In this example, two extraction columns wereconstructed: a 10 μL bed volume and 20 μL bed volume. To construct acolumn, various components were made by inserting the tips into severalcustom aluminum cutting tools and cutting the excess material extendingout of the tool with a razor blade to give specified column lengths anddiameters.

[0193] Referring to FIG. 7, the first cut 92 was made to the tip 92 of apipette tube 90 to form a 1.25 mm inside diameter hole 94 on the lowercolumn body, and a second cut 96 was made to form a lower column bodysegment 98 having a length of 15.0 mm.

[0194] Referring to FIG. 8, a cut 102 was made to the separate pipettetip 100 to form the upper column body 104. To make a 10 μL bed volumecolumn, the cut 102 was made to provide a tip 106 outside diameter of2.09 mm so that when the upper body was inserted into the lower body,the column height of the solid phase media bed 114 (FIG. 10) was 4.5 mm.To make a 20 μL bed volume column, the cut 102 was made to provide a tipoutside diameter of 2.55 mm cut so that when the upper body was insertedinto the lower body, the column height of the solid phase media bed 114(FIG. 10) was 7.0 mm.

[0195] Referring to FIG. 9, a 43 μm pore size Spectra/Mesh® polyestermesh material (Spectrum Labs, Ranch Dominguez, Calif., PN 145837) wascut into discs by a circular cutting tool (Pace Punches, Inc., Irvine,Calif.) and attached to the ends 106 and 108 upper column and lowercolumn bodies to form the membrane screens 110 and 112. The membranescreens were attached using PLASTIX® cyanoacrylate glue (Loctite, Inc.,Avon, Ohio). The glue was applied to the polypropylene body and thenpressed onto the membrane screen material. Using a razor blade, excessmesh material was removed around the outside perimeter of each columnbody end.

[0196] Referring to FIG. 10, the upper column body 104 is inserted intothe top of the lower column body segment 98 and pressed downward tocompact the solid phase media bed 114 to eliminate excess dead volumeabove the top of the bed.

Example 2

[0197] Preparation of SEPHAROSE™ Protein G and MEP HYPERCEL™ ExtractionColumns

[0198] Referring to FIG. 9, a suspension of Protein G SEPHAROSE™ 4 FastFlow, 45-165 μm particle size, (Amersham Biosciences, Piscataway, N.J.,PN 17-0618-01) in water/ethanol was prepared, and an appropriate amountof material 114 was pipetted into the lower column body 98.

[0199] Referring to FIG. 10, the upper column body 104 was pushed intothe lower column body 98 so that no dead space was left at the top ofthe bed 114, that is, at the top of the column bed. Care was taken sothat a seal was formed between the upper and lower column bodies 104 and98 while retaining the integrity of the membrane screen bonding to thecolumn bodies.

[0200] Several tips of 10 μL and 20 μL bed volumes were prepared.Several MEP (Mercapto-Ethyl-Pyridine) HYPERCEL™ (Ciphergen, Fremont,Calif., PN 12035-010) extraction columns were prepared using the sameprocedure. MEP HyperCel™ resin is a sorbent, 80-100 μm particle size,designed for the capture and purification of monoclonal and polyclonalantibodies. The extraction columns were stored with an aqueous solutionof 0.01% sodium azide in a refrigerator before use.

Example 3

[0201] Purification of Anti-leptin Monoclonal Antibody IgG with 10 μLand 20 μL Bed Volume Protein G SEPHAROSE™ Extraction Columns

[0202] A Protein G SEPHAROSE™ 4 Fast Flow (Amersham Biosciences,Piscataway, N.J., PN 17-0618-01) extraction column was prepared asdescribed in Example 2.

[0203] Five hundred μL serum-free media (HTS Biosystems, Hopkinton,Mass.) containing IgG (HTS Biosystems, Hopkinton, Mass.) of interest wascombined with 500 μL standard PBS buffer. The resulting 1 mL sample waspulled into the pipette tip, through the Protein G packed bed at a flowrate of approximately 1 mL/min) or roughly 15 cm/min). The sample wasthen pushed out to waste at the same approximate flow rate. Extraneousbuffer was removed from the bed by pulling 1 mL of deionized water intothe pipette column at about 1 mL/min and pushing it out at about 1mL/min. The water was pushed out as much as possible to achieve as dryof a column bed as is possible. The IgG was eluted from the column bedby drawing up an appropriate eluent volume of 100 mM glycine.HCl, pH 2.5(20 μL eluent in the case of a 20 μL bed volume, 15 μL eluent in thecase of a 10 μL bed volume). When the eluent was fully drawn into thebed, it was “pumped” back and forth through the bed five or six times,and the IgG-containing eluent was then fully expelled from the bed. Theeluted material was then neutralized with 100 mM NaH₂PO₄/100 mM Na₂HPO₄(5 μL neutralization buffer in the case of a 20 μL bed volume, 4 μLneutralization buffer in the case of a 10 μL bed volume). The purifiedand enriched antibodies were then ready for arraying.

Example 4

[0204] Purification of Anti-leptin Monoclonal Antibody IgG with 10 μLand 20 μL Bed Volume Protein G SEPHAROSE™ Extraction Columns

[0205] A Protein G SEPHAROSE™ 4 Fast Flow (Amersham Biosciences,Piscataway, N.J., PN 17-0618-01) extraction column was prepared asdescribed in Example 2.

[0206] Five hundred μL serum-free media (HTS Biosystems, Hopkinton,Mass.) containing IgG (HTS Biosystems, Hopkinton, Mass.) of interest wascombined with 500 μL standard PBS buffer. The resulting 1 mL sample waspulled into the pipette tip, through the Protein G packed bed at a flowrate of approximately 1 mL/min (or roughly 150 cm/min linear velocity).The sample was then pushed out to waste at the same approximate flowrate. Extraneous buffer was removed form the bed by pulling 1 mL ofdeionized water into the pipette column at about 1 mL/min and pushing itout at about 1 mL/min. The water was pushed out as much as possible toachieve as dry of a column bed as is possible. The IgG was eluted fromthe column bed by drawing up an appropriate eluent volume of 10 mMphosphoric acid (H₃PO₄), pH 2.5 (20 μL eluent in the case of a 20 μL bedvolume, 15 μL eluent in the case of a 10 μL bed volume). When the eluentwas fully drawn into the bed, it was “pumped” back and forth through thebed five or six times, and the IgG-containing eluent is then fullyexpelled from the bed. The eluted material was then neutralized with aspecially designed phosphate neutralizing buffer of 100 mM H₂NaPO₄/100mM HNa₂PO₄, pH 7.5 (5 μL neutralization buffer in the case of a 20 μLbed volume, 4 μL neutralization buffer in the case of a 10 μL bedvolume). The purified and enriched antibodies were then ready forarraying.

Example 5

[0207] Analysis of Purified IgG with Grating-coupled Surface PlasmonResonance (GC-SPR)

[0208] The anti-leptin monoclonal antibody IgG purified sample fromExample 4 was analyzed with GC-SPR. The system used for analysis was aFLEX CHIP™ Kinetic Analysis System (HTS Biosystems, Hopkinton, Mass.),which consists of a plastic optical grating coated with a thin layer ofgold on to which an array of biomolecules is immobilized. To immobilizethe purified IgG, the gold-coated grating was cleaned thoroughly withEtOH (10-20 seconds under a stream of ETOH). The gold-coated grating wasthen immersed in a 1 mM solution of 11-mercaptoundecanoic acid (MUA) inEtOH for 1 hour to allow for the formation of a self-assembledmonolayer. The surface was rinsed thoroughly with EtOH and ultra-purewater, and dried under a stream of nitrogen. A fresh solution of 75 mMEDC (1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide hydrochloride) and15 mM Sulfo-NHS (N-Hydroxysulfo-succinimide) was prepared in water. Analiquot of the EDC/NHS solution was delivered to the surface and allowedto react for 20-30 minutes, and the surface was then rinsed thoroughlywith ultra-pure water. An aliquot of 1 mg/mL Protein A/G in PBS, pH 7.4was delivered to the surface. The surface was placed in a humidenvironment and allowed to react for 1-2 hours. The surface was allowedto air dry, was rinsed with ultra-pure water and then dried under astream of nitrogen. Immediately prior to arraying of the IgGs, thesurface was rehydrated by placing in a humidified chamber, such asavailable with commercial arraying systems (e.g. Cartesian MicroSyssynQUAD System). The purified anti-leptin IgG was arrayed onto thesurface as described previously (J. Brockman, et al, “Grating-CoupledSPR: A Platform for Rapid, Label-free, Array-Based Affinity Screening ofFabs and Mabs”, 12^(th) Annual Antibody Engineering Conference, Dec.2-6, 2001, San Diego, Calif.) and the surface was introduced to the HTSBiosystems FLEX CHIP System. 150 nM leptin in PBS, pH 7.4 was introducedto the surface through the FLEX CHIP System, and real-time bindingsignals were collected as described previously (ibid.). These real-timebinding signals were mathematically processed in a manner describedpreviously (D. Myszka, “Kinetic analysis of macromolecular interactionsusing surface plasmon resonance biosensors”, Current Opinion inBiotechnology, 1997, Vol 8, pp. 50-57) for extraction of the associationrate (k_(a)), dissociation rate (k_(d)), and the dissociation affinityconstant (K_(d)=k_(d)/k_(a)). The kinetic data obtained is shown inTable II below. TABLE II Serum-free medium PBS No processing Mean K_(d)18 nM 3.2 nM (Adequate [IgG]) Starting [IgG] 500 μg/mL 500 μg/mL Withprocessing Mean K_(d) 6.6 nM 5.9 nM* (Insufficient [IgG] Starting [IgG]20 μg/mL 500 μg/mL*

[0209] The first set of data for “No processing” indicates that whensufficient IgG is present for detection (500 μg/mL) that theconstituents from the serum-free medium can contribute to inaccuracies.These data indicate for equal concentrations of IgG spotted within anexperiment, the calculated dissociation affinity constant can be nearlysix-fold different from one another (18 nM vs 3.2 nM). This can only bea result of components within the serum-free medium being co-arrayedwith the IgG, since the concentration and composition of IgG isidentical for each sample. Therefore, there is a demonstrated need forremoval of any extraneous components prior to arraying, which isindependent of IgG concentration.

[0210] The second set of data for “With processing” indicates that wheninsufficient IgG quantities are present for detection (20 μg/mL) thatsample processing not only allows for generation of sufficientprocessable signals, but also eliminates the inaccuracies generated fromextraneous components. These data indicate that the dissociationaffinity constants are virtually identical for 500 μg/mL purified IgG inPBS (unprocessed) as those calculated from 20 μg/mL IgG in serum-freemedium once processed with the current invention (5.9 nM vs 6.6 nM).

Example 6

[0211] Purification of Nucleic Acids with an Extraction Column

[0212] Columns from Example 1 are bonded with a 21 μm pore sizeSPECTRA/MESH® polyester mesh material (Spectrum Labs, Ranch Dominguez,Calif., PN 148244) by the same procedure as described in Example 2. A 10μL bed volume column is filled with PELLICULAR C18 (Alltech, Deerfield,Ill., PN 28551), particle size 30-50 μm. One end of the extractioncolumn is connected to a pipettor pump (Gilson, Middleton, Wis., P-1000PipetteMan) and the other end is movable and is connected to anapparatus where the materials may be taken up or deposited at differentlocations.

[0213] The extraction column consists of a 1 mL syringe (VWR, Brisbane,Calif., PN 53548-000), with one end connected to a pipettor pump(Gilson, Middleton, Wis., P-1000 PipetteMan) and the other end ismovable and is connected to an apparatus where the materials may betaken up or deposited at different locations.

[0214] A 100 μL sample containing 0.01 μg of DNA is prepared using PCRamplification of a 110 bp sequence spanning the allelic MstII site inthe human hemoglobin gene according to the procedure described in U.S.Pat. No. 4,683,195. A 10 μL concentrate of triethylammonium acetate(TEAA) is added so that the final volume of the solution is 110 μL andthe concentration of the TEAA in the sample is 100 mM. The sample isintroduced into the column and the DNA/TEAA ion pair complex isadsorbed.

[0215] The sample is blown out of the column and 10 μL of 50% (v/v)acetonitrile/water is passed through the column, desorbing the DNA, andthe sample is deposited into a vial for analysis.

Example 7

[0216] Desalting Proteins with an Extraction Column

[0217] Columns from Example 1 are bonded with a 21 μm pore sizeSPECTRA/MESH® polyester mesh material (Spectrum Labs, Ranch Dominguez,Calif., PN 148244) by the same procedure as described in Example 2. A 10μL bed volume column is filled with PELLICULAR C18 (Alltech, Deerfield,Ill., PN 28551), particle size 30-50 μm. One end of the extractioncolumn is connected to a pipettor pump (Gilson, Middleton, Wis., P-1000PipetteMan) and the other end is movable and is connected to anapparatus where the materials may be taken up or deposited at differentlocations.

[0218] The sample is a 100 μL solution containing 0.1 μg of Proteinkinase A in a phosphate buffer saline (0.9% w/v NaCl, 10 mM sodiumphosphate, pH 7.2) (PBS) buffer. Ten μL of 10% aqueous solution oftrifluoroacetic acid (TFA) is added so that the final volume of thesolution is 110 μL and the concentration of the TFA in the sample is0.1%. The sample is introduced into the column and the protein/TFAcomplex is adsorbed to the reverse phase of the bed.

[0219] The sample is blown out of the column and 10 μL of 50% (v/v)acetonitrile/water is passed through the column, desorbing the proteinfrom the bed of extraction media, and the sample is deposited into avial for analysis.

[0220] Alternatively, the bed may be washed with 10 μL of aqueous 0.1%TFA. This solution is ejected from the column and the protein isdesorbed and deposited into the vile.

[0221] If necessary, alternatively 1% heptafluorobutyric acid (HFBA) isused instead of TFA to reduce ion suppression effect when the sample isanalyzed by electrospray ion trap mass spectrometry.

Example 8

[0222] Straight Connection Configuration

[0223] This example describes an embodiment wherein the column body isconstructed by engaging upper tubular members and membrane screens in astraight configuration.

[0224] Referring to FIG. 11, the column consists of an upper tubularmember 120, a lower tubular member 122, a top membrane screen 124, abottom membrane screen 126, and a lower tubular circle 134 to hold thebottom membrane screen in place. The top membrane screen is held inplace by the upper and lower tubular members. The top membrane screen,bottom membrane screen and the channel surface 130 of the lower tubularmember define an extraction media chamber 128, which contains a bed ofextraction media 132 (i.e., packing material). The tubular members asdepicted in FIG. 11 are frustoconical in shape, but in relatedembodiments could take other shapes, e.g, cyclindrical.

[0225] To construct a column, various components are made by forminginjected molded members from polypropylene or machined members from PEEKpolymer to give specified column lengths and diameters and ends that canfit together, i.e., engage with one another. The configuration of themale and female portions of the column body is shaped differentlydepending on the method used to assemble the parts and the method usedto keep the parts together.

[0226] The components are glued or welded. Alternatively, they aresnapped together. In the case of snapping the pieces together, thefemale portion contains a lip and the male portion contains a ridge thatwill hold and seal the pieces once they are assembled. The membranescreen is either cut automatically during the assembly process or istrimmed after assembly.

Example 9

[0227] End Cap and Retainer Ring Configuration

[0228] This example describes an embodiment where an end cap andretainer ring configuration is used to retain the membrane screenscontaining a 20 μl bed of column packing material. The embodiment isdepicted in FIG. 12.

[0229] Referring to the figure, pipette tip 140 (VWR, Brisbane, Calif.,PN 53508-987) was cut with a razor blade to have a flat and straightbottom end 142 with the smooth sides such that a press fit can beperformed later. An end cap 144 was machined from PEEK polymer tubing tocontain the bottom membrane screen 146.

[0230] Two different diameter screens were cut from polyester mesh(Spectrum Labs, Ranch Dominguez, Calif., PN 145836) by a circularcutting tool (Pace Punches, Inc., Irving, Calif.), one for the topmembrane screen 148 and the other for the bottom membrane screen 146.The bottom membrane screen was placed into the end cap and pressed ontothe end of the cut pipette tip.

[0231] A 20 μL volume bed of beads 150 was formed by pipetting a 40 μLof 50% slurry of protein G agarose resin into the column body.

[0232] Two retainer rings were used to hold the membrane screen in placeon top of the bed of beads. The retainer rings were prepared by taking ⅛inch diameter polypropylene tubing and cutting thin circles from thetubing with a razor blade. A first retainer ring 152 was placed into thecolumn and pushed down to the top of the bed with a metal rod of similardiameter. The membrane screen 148 was placed on top of the firstretainer ring and then a second retainer ring 154 was pushed down to“sandwich” the membrane screen while at the same time pushing the wholescreen configuration to the top of the bed and ensuring that all deadvolume was removed. The membrane is flexible and naturally forms itselfto the top of the bed.

[0233] The column was connected to a 1000 μL pipettor (Gilson,Middleton, Wis., P-1000 PipetteMan) and water was pumped through the bedand dispensed from the bed. The column had low resistance to flow forwater solvent.

[0234] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover andvariations, uses, or adaptations of the invention that follow, ingeneral, the principles of the invention, including such departures fromthe present disclosure as come within known or customary practice withinthe art to which the invention pertains and as may be applied to theessential features hereinbefore set forth. Moreover, the fact thatcertain aspects of the invention are pointed out as preferredembodiments is not intended to in any way limit the invention to suchpreferred embodiments.

What is claimed is:
 1. A low dead volume extraction column comprising:i) a column body having an open upper end for attachment to a pump, anopen lower end for passing fluid into and out of the column body, and anopen channel between the upper and lower end of the column body; ii) abottom frit bonded to and extending across the open channel, the bottomfrit having a low pore volume; iii) a top frit bonded to and extendingacross the open channel between the bottom frit and the open upper endof the column body, the top frit having a low pore volume, wherein thetop frit, bottom frit, and channel surface define an extraction mediachamber; and iv) a bed of extraction media positioned inside theextraction media chamber.
 2. The low dead volume extraction column ofclaim 1, wherein the bottom frit is located at the open lower end of thecolumn body.
 3. The low dead volume extraction column of claim 1,wherein the bottom frit is less than 200 microns thick.
 4. The low deadvolume extraction column of claim 1, wherein the bottom frit has a porevolume equal to 10% or less of the interstitial volume of the bed ofextraction media.
 5. The low dead volume extraction column of claim 1,wherein the bottom frit has a pore volume of 0.5 microliters or less. 6.The low dead volume extraction column of claim 1, wherein the extractionmedia comprises a packed bed of gel-type packing material.
 7. The lowdead volume extraction column of claim 5, wherein the gel-type packingmaterial is selected from the group consisting of agarose and sepharose.8. The low dead volume extraction column of claim 1, wherein the bed ofextraction media has a bed volume of less than 20 microliters.
 9. Thelow dead volume extraction column of claim 1, wherein the bottom frit isa membrane screen and the top frit is optionally a membrane screen. 10.The low dead volume extraction column of claim 8, wherein membranescreen comprises a nylon or polyester woven membrane.
 11. The low deadvolume extraction column of claim 1, wherein the extraction mediacomprises a gel-type chromatography bead.
 12. The low dead volumeextraction column of claim 1, wherein the extraction media comprises anaffinity binding group having an affinity for a biological molecule ofinterest.
 13. The low dead volume extraction column of claim 11, whereinthe affinity binding group is elected from the group consisting ofProtein A, Protein G and an immobilized metal.
 14. The low dead volumeextraction column of claim 1, wherein at the column body comprises apolycarbonate, polypropylene or polyethylene material.
 15. The low deadvolume extraction column of claim 1, wherein the first and secondmembrane filters are bonded to the column body by gluing or welding. 16.The low dead volume extraction column of claim 1, wherein the volume ofthe extraction media chamber is less than 20 microliters.
 17. The lowdead volume extraction column of claim 1, wherein the bed of extractionmedia has a dry weight of less than 2 mgs.
 18. The low dead volumeextraction column of claim 1, wherein the extraction media comprise anextraction bead selected from the group consisting of affinity beadsused for protein purification, ion exchange beads used for proteinpurification, hydrophobic interaction beads used for proteinpurification, reverse phase beads used for nucleic acid or proteinpurification, agarose protein G beads used for IgG protein purification,and Hypercell beads used for IgG protein purification.
 19. The low deadvolume extraction column of claim 1, wherein the column body comprises aluer adapter, a syringe or a pipette tip.
 20. The low dead volumeextraction column of claim 1, wherein the upper end of the column bodyis attached to a pump for aspirating fluid through the lower end of thecolumn body.
 21. The low dead volume extraction column of claim 1,wherein the pump is a pipettor, a syringe, a peristaltic pump, anelectrokinetic pump, or an induction based fluidics pump.
 22. The lowdead volume extraction column of claim 1 comprising: i) a lower tubularmember comprising the lower end of the column body, a first engagingend, and a lower open channel between the lower end of the column bodyand the first engaging end; and ii) an upper tubular member comprisingthe upper end of the column body, a second engaging end, and an upperopen channel between the upper end of the column body and the secondengaging end, the top membrane screen of the extraction column bonded toand extending across the upper open channel at the second engaging end;wherein the first engaging end engages the second engaging end to form asealing engagement.
 23. The low dead volume extraction column of claim22, wherein the first engaging end has an inner diameter that matchesthe external diameter of the second engaging end, and wherein the firstengaging end receives the second engaging end in a telescoping relation.24. The low dead volume extraction column of claim 23, wherein the firstengaging end has a tapered bore that matches a tapered external surfaceof the second engaging end.
 25. A method for extracting an analyte froma sample solution comprising the steps of: i) contacting the lower endof the column body of the extraction column of claim 20 with a samplesolution containing an analyte and aspirating a quantity of the samplesolution into the column, whereby the quantity of sample solution entersthe bed of extraction media and the analyte is adsorbed by theextraction media; ii) discharging the sample solution out through thelower end of the extraction column body; iii) contacting the lower endof the column body with a desorption solvent and aspirating a quantityof the desorption solvent into the column, whereby the quantity ofsample desorption enters the bed of extraction media and the analyte isdesorbed from the extraction media into the desorption solvent; and iv)discharging the analyte-containing desorption solvent out through thelower end of the column.
 26. The method of claim 25, wherein the columnis attached to a pump for aspirating and discharging fluid through thelower end of the column body and the pump is used to discharge thesample solution and analyte-containing desorption solvent from theextraction column.
 27. The method of claim 25, wherein between steps(ii) and (iii) a quantity of wash fluid is aspirated into the columnthrough the lower end of the column and then discharged out through thelower end of the column, thereby washing the bed of extraction media.28. The method of claim 25, wherein the volume of desorption solventaspirated into the column is less than 3-fold greater the interstitialvolume of the packed bed of extraction beads.
 29. The method of claim25, wherein the quantity of desorption solvent is aspirated anddischarged from the column more than once.
 30. The method of claim 25,wherein the analyte is a biological macromolecule.
 31. The method ofclaim 30, wherein the biological macromolecule is a protein.
 32. Themethod of claim 31, wherein the analyte-containing desorption solvent isintroduced onto a protein chip.
 33. The method of claim 31, wherein theanalyte-containing desorption solvent is introduced into a massspectrometer.
 34. The method of claim 31, wherein the sample solution isa hybridoma cell culture supernatant.